Fluorescence correlation spectroscopy: Diagnostics for sparse molecules

Abstract

The robust glow of molecular fluorescence renders even sparse molecules detectable and susceptible to analysis for concentration, mobility, chemistry, and photophysics. Correlation spectroscopy, a statistical-physics-based tool, gleans quantitative information from the spontaneously fluctuating fluorescence signals obtained from small molecular ensembles. This analytical power is available for studying molecules present at minuscule concentrations in liquid solutions (less than one nanomolar), or even on the surfaces of living cells at less than one macromolecule per square micrometer. Indeed, routines are becoming common to detect, locate, and examine individual molecules under favorable conditions. The analytical strategy of fluorescence correlation spectroscopy (FCS) was introduced more than 25 years ago to measure chemical kinetics and the associated modulation of molecular diffusability by analysis of concentration fluctuations about the equilibrium of a small ensemble (≈103) of molecules (1). Because FCS has recently been resurrected (2) to meet several modern demands for analytical and physical sensitivity in biophysics, we discuss† some of the concepts and the modern potential of this method in chemical and biological physics.